Cloning And Expression Analysis Of Key Enzymes CAD And SAD Of Lignin Synthesis In Pyrus Bretschneideri Cv.Dangshan Su

Pyrus bretschneideri cv.Dangshan Su originated from Dangshan of Anhui province. It is well-known at home and abroad for characteristics such as large, thin skin, juicy fruit, meat, more nuclear small, sweet crisp. In recent years, the bad natural environment, variety degeneration and extensive field management, which cause its fruit stone cells content increased and fleshy coarsens, taste more slag. This affects seriously its edible quality and economic value. Dangshan Su fruit stone cells belongs to short sclereid, lignin monomer synthesis and polymerization in the cell wall deposition which cause lignification of part thin wall cell and secondary wall thickening to form stone cells. The development of stone cells is closely related to the synthesis, transfer, and deposition of lignin. Cinnamyl alcohol dehydrogenase (CAD) is a key enzyme in lignin biosynthesis, reducing CAD activity could reduce the synthesis of lignin monomer. Sinapyl alcohol dehydrogenase(SAD) is specific for the reduction of sinapyl aldehyde to sinapyl alcohol in lignin biosynthesis. This study shows the Dangshan Su fruit as material, cloning key enzyme genes of CAD and SAD of lignin synthesis of Dangshan Su fruit, processing bioinformatics analysis. Construction of prokaryotic expression vector used for SDS-PAGE analysis. Real-time fluorescence quantitative expression analysis is carried on different development periods of Dangshan Su. Construction of eukaryotic expression vector for further study of subcellular localization analysis. The main experimental results are as follows:1. Using RT-PCR and RACE technology, we get two full length of key enzymes gene CAD and SAD of lignin synthesis, respectively named PbCAD and PbSAD, accession number KF268003 and KF537783 respectively. The full length of PbCAD is 1283 bp, which codes 325 amino acid with opening reading frame 978 bp. The full length of PbSAD is 1399 bp, which codes 362 amino acid with opening reading frame 1089 bp.2. Bioinformatics analysis shows that the PbCAD protein isoelectric point is about 6.61, weak acid, about 35.6 kDa of molecular weight, no obvious transmembrane structure and signal peptide, characteristic of NADPH binding sites, with epimerase structural domain which belongs to oxidoreductase enzyme relying on the NADPH epimerase/dehydratase family. Isoelectric point of PbSAD protein is about 6.86. PbSAD protein shows weak acid, molecular weight of about 38.9 kDa, PbSAD containing ADH_N structural domain (alcohol dehydrogenase GroES-like domain, mainly catalytic sites of alcohol dehydrogenase) and ADH_zinc_N structural domain (zinc binding sites), the protein structure domain prove that PbSAD belongs to alcohol dehydrogenase protein.3. The prokaryotic expression results show construction of two prokaryotic expression vector are completed. SDS-PAGE electrophoresis of recombinant proteins show that we detect the expected molecular weight corresponding to the size of the recombinant protein bands, which show PbCAD and PbSAD were effectively expressed in Escherichia coli.4. Real time quantitative expression analysis is carried on different periods and parts of the Dangshan Su pear. The results show that following the development of Dangshan Su fruits, the expression level of PbCAD gene exhibits fall after rising first, the peak appear in 63d after flowering. The expression level of PbCAD gene of different parts is:close to the skin> fruit in the middle> close to the core. It show that the consistent trend with the previous studies of the stone cells and lignin,which indicate PbCAD gene is involved in the synthesis of lignin.5. Connecting PbCAD to expression vector pCAMBIA1301-EGFP. We succeed in constructing eukaryotic expression vector pCAMBIA1301-PbCAD, and then carry on study of subcellular localization.